Method of measuring blood component, sensor used in the method, and measuring device

ABSTRACT

The present invention provides a method of measuring a component in blood, by which an amount of the component can be corrected accurately by measuring a hematocrit (Hct) value of the blood with high accuracy and high reliability and also provides a sensor used in the method. The method of measuring a component in blood using a biosensor comprising a first electrode system including a first working electrode on which at least an oxidoreductase that acts upon the component and a mediator are provided and a first counter electrode and a second working electrode on which the mediator is not provided. The first working electrode and the first counter electrode are used for obtaining the amount of the component and the second working electrode and the first working electrode are used for obtaining the amount of the blood cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 13/972,389filed Aug. 21, 2013, which is a Continuation of application Ser. No.12/829,077 filed Jul. 1, 2010, which is a Division of application Ser.No. 10/578,275, filed May 5, 2006, which is a U.S. National Stage ofPCT/JP2004/018020, filed Dec. 3, 2004, which applications areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of measuring a bloodcomponent, a sensor used in the method, and a measuring device.

BACKGROUND ART

Conventionally, sensors for measuring a blood component have been usedfor clinical tests, self-measurement of blood glucose level bydiabetics, etc. The configuration of the sensor for measuring a bloodcomponent is such that, for example, a cover is disposed on aninsulating substrate having a working electrode and a counter electrodeon its surface with a spacer intervening between the cover and theinsulating substrate. On the working electrode and the counterelectrode, a reagent containing an oxidoreductase, a mediator (anelectron carrier), and the like is provided, thereby forming an analysisportion. The analysis portion communicates with one end of a channel forleading blood to the analysis portion. The other end of the channel isopen toward the outside of the sensor so as to serve as a blood supplyport. Blood component analysis (e.g., analysis of blood glucose level)using the sensor configured as above is carried out in the followingmanner, for example. First, the sensor is set in a dedicated measuringdevice (a meter). Then, a fingertip or the like is punctured with alancet to cause bleeding, and the blood supply port of the sensor isbrought into contact with the blood that has come out. The blood isdrawn into the channel of the sensor by capillary action and flowsthrough the channel to be led to the analysis portion where the bloodcomes into contact with the reagent. Then, a redox reaction occursbetween a component in the blood and the oxidoreductase so that acurrent flows via the mediator. The current is detected, and themeasuring device calculates an amount of the blood component based onthe detected current and displays the value obtained by the calculation.

In the above-described manner, the sensor can measure the bloodcomponent. However, since the obtained measured value might be affectedby a hematocrit (Hct), it might be necessary to measure a Hct value andthen correct the amount of the blood component based on this Hct valuein order to obtain an accurate measured value. For example, there hasbeen a sensor that corrects an amount of a blood component by measuringa Hct value by the use of two working electrodes and one referenceelectrode (see Patent Document 1). Other than this, there has been amethod in which a Hct value is measured using a mediator (see PatentDocument 2). However, the conventional technique has a problemconcerning the accuracy and the reliability of the measured Hct value sothat the amount of the blood component cannot be corrected sufficientlyand accurately.

-   Patent Document 1: JP 2003-501627 A-   Patent Document 2: Japanese Patent No. 3369183

DISCLOSURE OF INVENTION Problem to be Solved by the Invention

With the foregoing in mind, it is an object of the present invention toprovide a method of measuring a blood component, by which an amount ofthe blood component can be corrected sufficiently and accurately bymeasuring a Hct value with high accuracy and high reliability and alsoto provide a sensor used in the method and a measuring device.

Means for Solving Problem

In order to achieve the above object, the measurement method accordingto the present invention is a method of measuring a component in blood,including: causing a redox reaction between the component in the bloodand an oxidoreductase in the presence of a mediator; detecting anoxidation current or a reduction current caused through the redoxreaction by an electrode system; and calculating an amount of thecomponent based on a value of the detected current. The method furtherincludes measuring a Hct value of the blood and correcting the amount ofthe component using this Hct value. The measurement of the Hct valueincludes: providing an electrode system having a working electrode and acounter electrode; providing a mediator on the counter electrode but noton the working electrode; supplying the blood to the electrode system;applying a voltage to the electrode system in this state to cause anoxidation current or a reduction current to flow between the electrodes;detecting the oxidation current or the reduction current; andcalculating the Hct value based on a value of the detected current.

Furthermore, the sensor according to the present invention is a sensorfor measuring a component in blood by causing a redox reaction of thecomponent and detecting an oxidation current or a reduction currentcaused through the redox reaction by an electrode. The sensor includes:a first analysis portion including a first electrode system on which atleast an oxidoreductase that acts upon the component and a mediator areprovided; and a second analysis portion including a second electrodesystem that includes a working electrode and a counter electrode, inwhich a mediator is provided on the counter electrode but not on theworking electrode. In the first analysis portion, the component in theblood is measured by causing a redox reaction between the component andthe oxidoreductase in the presence of the mediator and detecting by thefirst electrode system an oxidation current or a reduction currentcaused to flow when a voltage is applied. On the other hand, in thesecond analysis portion, a Hct value of the blood is measured bysupplying the blood to the second electrode system, applying a voltageto the blood in this state to cause an oxidation current or a reductioncurrent to flow between the working electrode and the counter electrode,and detecting a value of the oxidation current or the reduction current.

The measuring device according to the present invention is a measuringdevice for measuring a component in blood, including: means for holdingthe sensor of the present invention; means for applying a voltage to thefirst electrode system of the sensor; means for detecting an oxidationcurrent or a reduction current flowing through the first electrodesystem; means for calculating an amount of the component from a value ofthe detected current; means for applying a voltage to the secondelectrode system of the sensor; means for detecting an oxidation currentor a reduction current flowing through the second electrode system; andmeans for calculating a Hct value of the blood from a value of thedetected current.

Effects of the Invention

As described above, the present invention is characterized by themeasurement of a Hct value. That is, by providing a mediator only on acounter electrode in the measurement of a Hct value, the currentreflecting the Hct value can be measured easily with high accuracy andhigh reliability. Thus, according to the measurement method, the sensor,and the measuring device of the present invention, the amount of theblood component can be corrected sufficiently and accurately because itis corrected based on the Hct value measured with high accuracy and highreliability. As a result, it is possible to obtain a highly accurate andhighly reliable corrected value of the amount of the blood component.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an exploded perspective view showing an example of a sensoraccording to the present invention.

FIG. 2 is a sectional view of the sensor.

FIG. 3 is a plan view of the sensor.

FIG. 4 is an exploded perspective view of another example of a sensoraccording to the present invention.

FIG. 5 is a sectional view of the sensor.

FIG. 6 is a plan view of the sensor.

FIG. 7A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 7B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 7C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 8A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 8B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 8C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 9A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 9B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 9C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 10A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 10B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 10C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 11A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 11B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 11C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 12A shows how a reagent layer is provided in still another exampleof a sensor according to the present invention; FIG. 12B is a graphshowing changes in response current (μA) obtained in Hct measurementover time during voltage application in the example; and FIG. 12C is agraph showing changes in difference in sensitivity (%) over time duringthe voltage application in the example.

FIG. 13A shows how a reagent layer is provided in a sensor according toa comparative example; FIG. 13B is a graph showing changes in responsecurrent (μA) obtained in Hct measurement over time during voltageapplication in the comparative example; and FIG. 13C is a graph showingchanges in difference in sensitivity (%) over time during the voltageapplication in the comparative example.

FIG. 14A shows how a reagent layer is provided in a sensor according toanother comparative example; FIG. 14B is a graph showing changes inresponse current (μA) obtained in Hct measurement over time duringvoltage application in the comparative example; and FIG. 14C is a graphshowing changes in difference in sensitivity (%) over time during thevoltage application in the comparative example.

FIG. 15A shows how a reagent layer is provided in a sensor according tostill another comparative example; FIG. 15B is a graph showing changesin response current (μA) obtained in Hct measurement over time duringvoltage application in the comparative example; and FIG. 15C is a graphshowing changes in difference in sensitivity (%) over time during thevoltage application in the comparative example.

FIG. 16A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (0.5 V) in stillanother example of a sensor according to the present invention; and FIG.16B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 17A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (1.0 V) in stillanother example of a sensor according to the present invention; and FIG.17B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 18A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (1.5 V) in stillanother example of a sensor according to the present invention; and FIG.18B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 19A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (2.0 V) in stillanother example of a sensor according to the present invention; and FIG.19B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 20A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (2.5 V) in stillanother example of a sensor according to the present invention; and FIG.20B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 21A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (3.0 V) in stillanother example of a sensor according to the present invention; and FIG.21B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 22A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (3.5 V) in stillanother example of a sensor according to the present invention; and FIG.22B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 23A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (4.0 V) in stillanother example of a sensor according to the present invention; and FIG.23B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 24A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (4.5 V) in stillanother example of a sensor according to the present invention; and FIG.24B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 25A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (5.0 V) in stillanother example of a sensor according to the present invention; and FIG.25B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 26A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (5.5 V) in stillanother example of a sensor according to the present invention; and FIG.26B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 27A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (6.0 V) in stillanother example of a sensor according to the present invention; and FIG.27B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 28A is a graph showing changes in response current (μA) obtained inHct measurement over time during voltage application (6.5 V) in stillanother example of a sensor according to the present invention; and FIG.28B is a graph showing changes in difference in sensitivity (%) overtime during the voltage application in the example.

FIG. 29 is a perspective view showing an example of a measuring deviceaccording to the present invention.

FIG. 30 is a plan view showing still another example of a sensoraccording to the present invention.

FIG. 31 is a plan view showing the configuration of the measuring deviceaccording to the above example.

EXPLANATION OF REFERENCE NUMERALS

11, 12, 13, 21, 22, 23, 24, 81, 82, 111, 112, 113, 114 electrode

14, 25, 83 reagent portion (reagent layer)

15, 26, 84 channel

16, 27, 85 air vent hole

101, 201, 801 insulating substrate

102, 202, 802 spacer

103, 203, 803 cover

121 sensor

122 sample supply port

130, 123 measuring device

124 display portion

125 attachment portion

131 CPU

132 LCD

133 reference voltage source

134 A/D conversion circuit

135 current/voltage conversion circuit

136 switching circuit

137 a, 137 b, 137 c, 137 d connector

DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail.

In the method of measuring a blood component and the sensor according tothe present invention, the mediator used for the Hct measurement or inthe second analysis portion is not particularly limited. Examples of themediator include a ferricyanide, p-benzoquinone, p-benzoquinonederivatives, phenazine methosulfate, methylene blue, ferrocene, andferrocene derivatives. Among these, a ferricyanide is preferable, andpotassium ferricyanide is more preferable. The amount of the mediator tobe blended is not particularly limited, but is, for example, 0.1 to 1000mM, preferably 1 to 500 mM, and more preferably 10 to 200 mM per onemeasurement or one sensor.

In the method of measuring a blood component and the sensor according tothe present invention, the electrode that is used for the Hctmeasurement or in the second analysis portion and on which the mediatoris not provided preferably is coated with a polymeric material in orderto prevent adhesion of impurities, oxidation of the electrode, and thelike. Examples of the polymeric material include carboxymethyl cellulose(CMC), hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxyethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyamino acid suchas polylysine, polystyrene sulfonate, gelatin and derivatives thereof,polyacrylic acid and salts thereof, polymethacrylic acid and saltsthereof, starch and derivatives thereof, maleic anhydride polymer andsalts thereof, and agarose gel and derivatives thereof. They may be usedindividually or two or more of them may be used together. The method ofcoating the electrode with a polymeric material is not particularlylimited. For example, the coating can be achieved by providing apolymeric material solution, applying the solution to the electrodesurface, and then removing a solvent contained in the coating layer ofthe solution by drying.

In the method of measuring a blood component and the sensor according tothe present invention, a voltage applied between the working electrodeand the counter electrode that are used for the Hct measurement or inthe second analysis portion preferably is equal to or higher than avoltage causing electrolysis of water, more preferably in the range from1 to 10 V, and still more preferably in the range from 1 to 6.5 V. Byapplying a voltage that is equal to or higher than a voltage causingelectrolysis of water, a current depending on a hematocrit alone can bemeasured with a still higher sensitivity. As a result, it is possible toobtain a stable current that is not affected by other redox substancespresent in blood and thus does not vary depending on a specimen (anindividual). The voltage is applied for, for example, 0.001 to 60seconds, preferably 0.01 to 10 seconds, and more preferably 0.01 to 5seconds.

In the method of measuring a blood component and the sensor according tothe present invention, it is preferable that the shortest distancebetween the working electrode and the counter electrode that are usedfor the Hct measurement or in the second analysis portion is at least0.05 mm. When the distance between the electrodes is at least 0.05 mm asdescribed above, the reliability of the measured value is improved. Morepreferably, the distance between the electrodes is at least 0.1 mm,still more preferably at least 0.5 mm.

In the method of measuring a blood component according to the presentinvention, the correction using the Hct value preferably is carried outbased on a previously prepared calibration curve or calibration tablefor showing the relationship between a Hct value and an amount of theblood component.

In the method of measuring a blood component according to the presentinvention, the order of carrying out the blood component measurement andthe Hct measurement is not particularly limited. However, in the casewhere the same electrode is used in both the measurements as will bedescribed later, it is preferable that the blood component is measuredfirst and the Hct value is measured thereafter. Note here that the casewhere the electrode that is used as a working electrode in the bloodcomponent measurement is used as a counter electrode in the Hctmeasurement corresponds to the above case. On this electrode, a mediator(e.g., potassium ferricyanide) that initially is in an oxidized state isprovided. This mediator is reduced through the enzyme reaction caused inthe blood component measurement and is oxidized again for the purpose ofmeasuring the blood component. Thus, after the blood componentmeasurement, ferricyanide ions are present dominantly at the interfaceof the electrode. On the other hand, it is preferable that a largeamount of ferricyanide ions are present in the vicinity of a counterelectrode used for the Hct measurement in order to suppress anelectrolytic reduction reaction occurring at the counter electrode frombeing a rate-determining step. On this account, it is preferable thatthe electrode used as a working electrode in the blood componentmeasurement is used as a counter electrode in the Hct measurement afterthe completion of the blood component measurement.

In the method of measuring a blood component according to the presentinvention, it is preferable that the electrode system for detecting theoxidation current or the reduction current in the measurement of theblood component includes a working electrode and a counter electrode.

Preferably, the method of measuring a blood component according to thepresent invention further includes measuring a temperature of ameasurement environment, and the amount of the blood component iscorrected using the measured temperature. This is because the enzymereaction is affected by the temperature of the measurement environment.In this case, it is preferable that the correction using the temperatureis carried out based on a previously prepared calibration curve orcalibration table.

In the method of measuring a blood component and the sensor according tothe present invention, the blood component to be measured is, forexample, glucose, lactic acid, uric acid, bilirubin, cholesterol, or thelike. Furthermore, the oxidoreductase is selected as appropriatedepending on the blood component to be measured. Examples of theoxidoreductase include glucose oxidase, lactate oxidase, cholesteroloxidase, bilirubin oxidase, glucose dehydrogenase, and lactatedehydrogenase. The amount of the oxidoreductase is, for example, 0.01 to100 U, preferably 0.05 to 10 U, and more preferably 0.1 to 5 U per onesensor or one measurement. Among these, the blood component to bemeasured preferably is glucose, and the oxidoreductase to be used inthis case preferably is glucose oxidase or glucose dehydrogenase.

In the sensor for measuring a blood component according to the presentinvention, it is preferable that the first electrode system includes aworking electrode and a counter electrode. Furthermore, in the sensor ofthe present invention, it is preferable that, in the first electrodesystem and the second electrode system, at least one of the electrodesor all the electrodes provided in the first electrode system also serveas the counter electrode in the second electrode system. It is morepreferable that, in the first electrode system and the second electrodesystem, only the working electrode in the first electrode system alsoserves as the counter electrode in the second electrode system.

In the sensor for measuring a blood component according to the presentinvention, the mediator provided on the first electrode system is notparticularly limited, and examples thereof include a ferricyanide,p-benzoquinone, p-benzoquinone derivatives, phenazine methosulfate,methylene blue, ferrocene, and ferrocene derivatives. Among these, aferricyanide is preferable, and potassium ferricyanide is morepreferable. The amount of the mediator to be blended is not particularlylimited, but is, for example, 0.1 to 1000 mM, preferably 1 to 500 mM,and more preferably 10 to 200 mM per one measurement or one sensor.

The sensor for measuring a blood component according to the presentinvention preferably is configured so that it further includes aninsulating substrate, the first analysis portion, the second analysisportion, and a channel for leading the blood to the analysis portionsare formed on the insulating substrate, and one end of the channel isopen toward the outside of the sensor so as to serve as a blood supplyport. In this case, the sensor may be configured so that there is onlyone blood supply port and the channel branches so that ends of branchedportions communicate with the analysis portions, respectively.Alternatively, the sensor may be configured so that the second analysisportion is located in the channel and the first analysis portion islocated farther from the blood supply port than the second analysisportion.

Preferably, the sensor for measuring a blood component according to thepresent invention is configured so that it further includes a spacer anda cover and the cover is disposed on the insulating substrate via thespacer.

In the sensor for measuring a blood component according to the presentinvention, it is preferable that a polymeric material, an enzymestabilizer, and a crystal homogenizing agent further are provided on thefirst electrode system.

The polymeric material serves to prevent adhesion of impurities to theelectrode surface and oxidation of the electrode surface as well as toprotect the electrode surface. Examples of the polymeric materialinclude CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, ethyl hydroxyethyl cellulose, carboxyethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyamino acid suchas polylysine, polystyrene sulfonate, gelatin and derivatives thereof,polyacrylic acid and salts thereof, polymethacrylic acid and saltsthereof, starch and derivatives thereof, maleic anhydride polymer andsalts thereof, and agarose gel and derivatives thereof. They may be usedindividually or two or more of them may be used together. Among these,CMC is preferable. The ratio of the polymeric material to an entirereagent solution for preparing a reagent portion is, for example, 0.001to 10 wt %, preferably 0.005 to 5 wt %, and more preferably 0.01 to 2 wt%.

As the enzyme stabilizer, sugar alcohol can be used, for example.Examples of the sugar alcohol include chain polyhydric alcohols andcyclic sugar alcohols, such as sorbitol, maltitol, xylitol, mannitol,lactitol, reduced paratinose, arabinitol, glycerol, ribitol, galactitol,sedoheptitol, perseitol, volemitol, styracitol, polygalitol, iditol,talitol, allitol, isylitol, hydrogenated glucose syrup, and isylitol.Note here that stereoisomers, substitution products, and derivatives ofthese sugar alcohols also may be used as the enzyme stabilizer. Thesesugar alcohols may be used individually or two or more of them may beused together. Among these, maltitol is preferable. The amount of theenzyme stabilizer to be blended is, for example, in the range from 0.1to 500 mM, preferably from 0.5 to 100 mM, and more preferably from 1 to50 mM per one measurement or one sensor.

The crystal homogenizing agent serves to homogenize the crystalcondition of the reagent portion. As the crystal homogenizing agent, anamino acid can be used, for example. Examples of the amino acid includeglycine, alanine, valine, leucine, isoleucine, serine, threonine,methionine, asparagine, glutamine, arginine, lysine, histidine,phenylalanine, tryptophan, proline, sarcosine, betaine, taurine, andsalts, substitution products, and derivatives of these amino acids. Theymay be used individually or two or more of them may be used together.Among these, glycine, serine, proline, threonine, lysine, and taurineare preferable, and taurine is more preferable. The amount of thecrystal homogenizing agent to be blended is, for example, 0.1 to 1000mM, preferably 10 to 500 mM, and more preferably 20 to 200 mM per onemeasurement or one sensor.

Preferably, the sensor for measuring a blood component according to thepresent invention is configured so that it further includes a blooddetecting electrode, and the blood detecting electrode is locatedfarther from the blood supply port than at least one of the analysisportions so that whether or not blood is supplied surely to the at leastone of the analysis portions can be detected by the blood detectingelectrode. It is more preferable that the blood detecting electrode islocated farther from the blood supply port than both the analysisportions.

Next, the measuring device according to the present invention preferablyfurther includes means for correcting the amount of the blood componentusing the Hct value. Furthermore, in the measuring device of the presentinvention, the voltage applied to the second electrode system preferablyis equal to or higher than a voltage causing electrolysis of water, morepreferably in the range from 1 to 10 V, and still more preferably from 1to 6.5 V.

FIG. 29 is a perspective view showing an example of a measuring deviceaccording to the present invention to which a sensor according to thepresent invention is attached. As shown in FIG. 29, this measuringdevice 123 has a sensor attachment portion 125 at one end, and a sensor121 is attached to this portion so as to be held by the measuringdevice. The reference numeral 122 denotes a sample supply port of thesensor 121. This measuring device 123 has a display portion 124 at asubstantially center portion thereof, and the result of the measurementis displayed in this display portion 124.

The measuring device according to the present invention preferablyincludes a connector, a switching circuit, a current/voltage conversioncircuit, an A/D conversion circuit, a reference voltage source, a CPU,and a liquid crystal display portion (LCD). By providing thesecomponents, the following operations become possible: applying a voltageto the first electrode system and the second electrode system in thesensor of the present invention; detecting the value of a currentflowing between these electrode systems; calculating an amount of theblood component or a Hct value based on the thus-detected current value;correcting the amount of the blood component based on the Hct value; anddisplaying the thus-obtained corrected value. With regard to the circuitconfiguration of a measuring device according to the present invention,an example thereof will be described later.

Hereinafter, examples of a sensor for measuring a blood componentaccording to the present invention will be described with reference tothe drawings.

EXAMPLE 1

FIGS. 1, 2, and 3 show one example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 1 is an explodedperspective view of the sensor, FIG. 2 is a sectional view of thesensor, and FIG. 3 is a plan view of the sensor. In these threedrawings, the same components are given the same reference numerals.

As shown in the drawings, in this sensor, three electrodes 11, 12, and13 are formed on an insulating substrate 101. Each of the electrodes canbe switched between a working electrode and a counter electrode. Thesurface of the electrode 13 is coated with a polymeric material such asCMC. On an electrode portion formed by the electrodes 11 and 12, areagent layer 14 is disposed. The reagent layer 14 contains anoxidoreductase such as glucose dehydrogenase and a mediator, andoptionally contains a polymeric material, an enzyme stabilizer, and acrystal homogenizing agent. The type and the blending ratio of thesereagents are as described above. A cover 103 is disposed on theinsulating substrate 101 so as to cover an entire area excluding one endportion (the end portion on the right in the drawings) with a spacer 102intervening therebetween. This sensor has a channel 15 for leading bloodto the electrode 13 and the electrodes 11 and 12. This channel 15branches into two portions so that the channel as a whole forms aT-shape, and ends of the branched portions communicate with theelectrode portions, respectively. The channel extends to the other endportion (the end portion on the left in the drawings) of the sensor andthe tip thereof is open toward the outside of the sensor so as to serveas a blood supply port. The three electrodes 11, 12, and 13 areconnected to leads, respectively. These leads extend to theabove-described one end portion of the sensor with the tip of each leadnot being covered with the cover but being exposed. The cover 103 hastwo air vent holes 16 at portions corresponding to the ends of thebranched portions of the channel 15.

In the present invention, the material of the insulating substrate isnot particularly limited, and may be, for example, polyethyleneterephthalate (PET), polycarbonate (PC), polyimide (PI), polyethylene(PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC),polyoxymethylene (POM), monomer-cast nylon (MC), polybutyleneterephthalate (PBT), polymethyl methacrylate (PMMA), an ABS resin (ABS),or glass. Among these, polyethylene terephthalate (PET), polycarbonate(PC), and polyimide (PI) are preferable, and polyethylene terephthalate(PET) is more preferable. The size of the insulating substrate is notparticularly limited. For example, the insulating substrate may have anoverall length of 5 to 100 m, a width of 2 to 50 mm, and a thickness of0.05 to 2 mm; preferably an overall length of 7 to 50 mm, a width of 3to 20 mm, and a thickness of 0.1 to 1 mm; and more preferably an overalllength of 10 to 30 mm, a width of 3 to 10 mm, and a thickness of 0.1 to0.6 mm.

The electrodes and the leads on the insulating substrate may be formedby, for example, forming a conductive layer with gold, platinum,palladium, or the like by sputtering or vapor deposition and thenprocessing the conductive layer into a particular electrode pattern witha laser. Examples of the laser include YAG lasers, CO₂ lasers, andexcimer lasers. Note here that the electrode pattern is not limited tothose shown in the examples or the like, and there is no limitationregarding the electrode pattern as long as it can achieve the effect ofthe present invention. The coating of the surface of the electrode 13can be achieved by, for example, preparing a solution of the polymericmaterial, dropping or applying this solution with respect to theelectrode surface, and then drying it. The drying may be, for example,natural drying, air drying, hot air drying, or heat drying.

The reagent portion 14 can be formed in the following manner, forexample. First, 0.1 to 5.0 U/sensor of PQQ-GDH, 10 to 200 mM ofpotassium ferricyanide, 1 to 50 mM of maltitol, and 20 to 200 mM oftaurine are dissolved in a 0.01 to 2.0 wt % CMC aqueous solution toprepare a reagent solution. The reagent solution is dropped on theelectrodes 11 and 12 formed on the substrate and then is dried, thusforming the reagent portion 14. The drying may be natural drying orforced drying using warm air, for example. However, if the temperatureof the warm air is too high, there is a possibility that the enzymecontained in the solution might be deactivated. Thus, the temperature ofthe warm air preferably is around 50° C.

In the present invention, the material of the spacer is not particularlylimited. For example, the same material as that of the insulatingsubstrate can be used. The size of the spacer also is not particularlylimited. For example, the spacer may have an overall length of 5 to 100mm, a width of 2 to 50 mm, and a thickness of 0.01 to 1 mm; preferablyan overall length of 7 to 50 mm, a width of 3 to 20 mm, and a thickness0.05 to 0.5 mm; and more preferably an overall length of 10 to 30 mm, awidth of 3 to 10 mm, and a thickness of 0.05 to 0.25 mm. The spacer hasa T-shaped cut-away portion that serves as the channel for leadingblood. The size of the cut-away portion is as follows, for example: thelength from the blood supply port to the branching part is 0.5 to 20 mm,the length from the branching part to the end of the branched portion is1 to 25 mm, and the width is 0.1 to 5 mm; preferably the length from theblood supply port to the branching part is 1 to 10 mm, the length fromthe branching part to the end of the branched portion is 1.5 to 10 mm,and the width is 0.2 to 3 mm; and more preferably the length from theblood supply port to the branching part is 1 to 5 mm, the length fromthe branching part to the end of the branched portion is 1.5 to 5 mm,and the width is 0.5 to 2 mm. The cut-away portion may be formed, forinstance, by using a laser, a drill, or the like, or by forming thespacer using a die that can form the spacer provided with the cut-awayportion.

In the present invention, the material of the cover is not particularlylimited. For example, the same material as that of the insulatingsubstrate can be used. It is more preferable that a portion of the covercorresponding to the ceiling of the sample supply channel is subjectedto a treatment for imparting hydrophilicity. The treatment for impartinghydrophilicity may be carried out by, for example, applying a surfactantor introducing a hydrophilic functional group such as a hydroxyl group,a carbonyl group, or a carboxyl group to the surface of the cover byplasma processing or the like. The size of the cover is not particularlylimited. For example, the cover may have an overall length of 5 to 100mm, a width of 3 to 50 mm, and a thickness of 0.01 to 0.5 mm; preferablyan overall length of 10 to 50 mm, a width of 3 to 20 mm, and a thicknessof 0.05 to 0.25 mm; and more preferably an overall length of 15 to 30mm, a width of 5 to 10 mm, and a thickness of 0.05 to 0.2 mm. The coverpreferably has an air vent hole. The shape of the air vent hole may be,for example, circular, oval, polygonal, or the like, and the maximumdiameter thereof may be, for example, 0.01 to 10 mm, preferably 0.025 to5 mm, and more preferably 0.025 to 2 mm. The cover may have a pluralityof air vent holes. The air vent hole may be formed, for instance, byperforating the cover with a laser, a drill, or the like, or by formingthe cover using a die that can form the cover provided with the air venthole. Then, by laminating the insulating substrate, the spacer, and thecover in this order and integrating them, the sensor can be obtained.The integration can be achieved by adhering these three components withan adhesive or through heat-sealing. As the adhesive, an epoxy adhesive,an acrylic adhesive, a polyurethane adhesive, a thermosetting adhesive(a hot melt adhesive or the like), a UV curable adhesive, or the likecan be used, for example.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

A voltage is applied between the electrode 11 and the electrode 13, andwhether or not the blood is supplied to the sensor is detected bydetecting the change in current accompanying the supply of the blood.After the supply of the blood has been confirmed, the subsequent step isstarted. Note here that the voltage applied in Step 1 is 0.05 to 1 V,for example.

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with the glucoseoxidoreductase for a certain period of time, a voltage is appliedbetween the electrode 11 as a working electrode and the electrode 12 asa counter electrode, thereby oxidizing a reduced mediator generated onthe electrode 11 through the enzyme reaction. The oxidation currentcaused at this time is detected. The glucose is allowed to react withthe oxidoreductase for, for example, 0 to 60 seconds, preferably 0.5 to30 seconds, and more preferably 1 to 10 seconds. In Step 2, the voltageapplied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V, and morepreferably 0.2 to 0.5 V, and the voltage application time is, forexample, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Hct Value)

By applying a voltage between the electrode 13 as a working electrodeand the electrode 11 as a counter electrode, a current depending on aHct value can be detected based on an electrolytic oxidation reaction ofblood components. Note here that the detected current can be convertedinto a Hct value using a previously prepared calibration curve orcalibration curve table. In this correction, a Hct value determinedusing a previously prepared calibration curve showing the relationshipbetween a current and a Hct value may be used or alternatively, thedetected current may be used as it is. In Step 3, the voltage appliedis, for example, 1 to 10 V, preferably 1 to 6.5 V, and more preferably 2to 3 V, and the voltage application time is, for example, 0.001 to 60seconds, preferably 0.01 to 10 seconds, and more preferably 0.01 to 5seconds. In Step 3, the oxidation current depending on a Hct value canbe detected without being affected by any reagent because no mediator isprovided on the electrode 13 as a working electrode, and the electrode13 and the electrode 11 are spaced apart from each other by a certaindistance with no reagent such as a mediator being provided in this spaceso that only blood is present in this space. Preferably, Step 3 isperformed after the completion of Step 2. Although the electrode 11 isused as a counter electrode in the present example, the measurement alsocan be achieved when the electrode 12 is used as a counter electrode.Also, it is possible to use both the electrodes 11 and 12 as counterelectrodes. Note here that when the surface of the electrode 13 is notcoated with a polymeric material or the like, it is still possible tocarry out the measurement.

(Step 4: Correcting Blood Component)

The amount of glucose obtained in Step 2 is corrected using the Hctvalue detected in Step 3. The correction preferably is carried out basedon a calibration curve (including a calibration table) preparedpreviously. The corrected amount of glucose is displayed on or stored inthe measuring device. Instead of determining the Hct value and thencorrecting the amount of glucose as described above, the currentdepending on the Hct value, which has been detected in Step 3, may beused as it is to correct the amount of glucose.

EXAMPLE 2

FIGS. 4, 5, and 6 show another example of a sensor for measuring a bloodcomponent according to the present invention. FIG. 4 is an explodedperspective view of the sensor, FIG. 5 is a sectional view of thesensor, and FIG. 6 is a plan view of the sensor. In these threedrawings, the same components are given the same reference numerals.

As shown in the drawings, in this sensor, four electrodes 21, 22, 23,and 24 are formed on an insulating substrate 201. These electrodes canbe switched between a working electrode and a counter electrode. Thesurface of the electrode 24 is coated with a polymeric material in themanner as described above. On an electrode portion formed by theelectrodes 21, 22, and 23, a reagent layer 25 is provided. The reagentlayer 25 contains an oxidoreductase such as glucose dehydrogenase and amediator, and optionally contains a polymeric material, an enzymestabilizer, and a crystal homogenizing agent. The type and the blendingratio of these reagents are as described above. A cover 203 is disposedon the insulating substrate 201 so as to cover an entire area excludingone end portion (the end portion on the right in the drawings) with aspacer 202 intervening therebetween. This sensor has a channel 26 forleading blood to the reagent portion 25. This channel 26 extendslinearly (I-shape). The channel 26 extends to the other end portion (theend portion on the left in the drawings) of the sensor and the tipthereof is open toward the outside of the sensor so as to serve as ablood supply port. The four electrodes are arranged in series in thechannel, and the electrode 22 is located farthest from the blood supplyport. The four electrodes 21, 22, 23, and 24 are connected to leads,respectively. These leads extend to the above-described one end portionof the sensor with the tip of each lead not being covered with the coverbut being exposed. The cover 203 has an air vent hole 27 at a portioncorresponding to the rear side of the channel 26.

In the present example, the material, the size, and the like of theinsulating substrate are not particularly limited, and may be the sameas in Example 1. Furthermore, the electrodes, the leads, the manner ofcoating the electrode surface with a polymeric material, and the reagentportion also are the same as in Example 1. Still further, the materialand the size of the spacer and the method of processing the spacer alsoare the same as in Example 1. In the present example, the spacer has anI-shaped cut-away portion that serves as the channel for leading blood.The size of the cut-away portion is as follows, for example: the overalllength is 0.5 to 50 mm and the width is 0.1 to 5 mm; preferably theoverall length is 1 to 10 mm and the width is 0.2 to 3 mm; and morepreferably the overall length is 1 to 5 mm and the width is 0.5 to 2 mm.The cut-away portion may be formed, for instance, by using a laser, adrill, or the like, or by forming the spacer using a die that can formthe spacer provided with the cut-away portion. The material and the sizeof the cover, the treatment for imparting hydrophilicity to the cover,and the air vent hole provided in the cover are the same as inExample 1. Also, the method for producing the sensor of the presentexample is the same as that for producing the sensor of Example 1.

Measurement of a blood glucose level using this sensor can be carriedout in the following manner, for example. First, a fingertip or the likeis punctured with a dedicated lancet to cause bleeding. On the otherhand, the sensor is set in a dedicated measuring device (a meter). Theblood supply port of the sensor set in the measuring device is broughtinto contact with the blood that has come out, so that the blood is ledinside the sensor by capillary action. Then, the sensor analyzes theblood according to the following steps.

(Step 1: Detecting Specimen (Blood))

Whether or not the blood is supplied to the end of the channel isdetected by applying a voltage between the electrode 24 and theelectrode 22. After the supply of the blood to the end of the channelhas been confirmed, the subsequent step is started. In the case wherethe blood is not supplied to the end of the channel, the measuringdevice recognizes it as the lack of the amount of the specimen anddisplays an error message. The voltage applied in Step 1 is, forexample, 0.05 to 1 V. In this case, the specimen can be detected bydetecting the change in current between the electrode 22 and any one ofother electrodes (21, 23, and 24).

(Step 2: Measuring Glucose)

After allowing glucose in the blood to react with the glucoseoxidoreductase for a certain period of time, a voltage is appliedbetween the electrode 21 as a working electrode and the electrode 23 asa counter electrode, thereby oxidizing a reduced mediator generated onthe electrode 21 through the enzyme reaction. The oxidation currentcaused at this time is detected. The glucose is allowed to react withthe oxidoreductase for, for example, 0 to 60 seconds, preferably 0.5 to30 seconds, and more preferably 1 to 10 seconds. In Step 2, the voltageapplied is, for example, 0.05 to 1 V, preferably 0.1 to 0.8 V, and morepreferably 0.2 to 0.5 V, and the voltage application time is, forexample, 0.01 to 30 seconds, preferably 0.1 to 10 seconds, and morepreferably 1 to 5 seconds.

(Step 3: Measuring Hct Value)

By applying a voltage between the electrode 24 as a working electrodeand the electrode 21 as a counter electrode, a current depending on aHct value can be detected. Based on the detected current, the Hct valueof the blood can be determined. The thus-determined Hct value is usedfor the correction in the measurement of glucose. In this correction, aHct value determined using a previously prepared calibration curveshowing the relationship between a current and a Hct value may be usedor alternatively, the detected current may be used as it is. In Step 3,the voltage applied is, for example, 1 to 10 V, preferably 1 to 6.5 V,and more preferably 2 to 3 V, and the voltage application time is, forexample, 0.001 to 60 seconds, preferably 0.01 to 10 seconds, and morepreferably 0.01 to 5 seconds. In Step 3, the oxidation current dependingon a Hct value can be detected without being affected by any reagentbecause no mediator is provided on the electrode 24 as a workingelectrode, and the electrode 24 and the electrode 21 are spaced apartfrom each other by a certain distance with no reagent such as a mediatorbeing provided in this space so that only blood is present in thisspace. Preferably, Step 3 is performed after the completion of Step 2.Although the electrode 21 alone is used as the counter electrode in thepresent example, the present invention is not limited thereto. It shouldbe noted that the electrode 23 alone, the electrode 22 alone, thecombination of the electrode 21 and the electrode 22, the combination ofthe electrode 21 and the electrode 23, the combination of the electrode22 and the electrode 23, the combination of the electrode 21, theelectrode 22, and the electrode 23 also may be used as the counterelectrode. Also, it should be noted that when the surface of theelectrode 13 is not coated with a polymeric material or the like, it isstill possible to achieve the measurement.

(Step 4: Correcting Blood Component)

The amount of glucose obtained in Step 2 is corrected using the Hctvalue detected in Step 3. The correction preferably is carried out basedon a calibration curve (including a calibration table) preparedpreviously. The corrected amount of glucose is displayed on or stored inthe measuring device.

EXAMPLE 3

In the present example, six types of sensors (3-1 to 3-6) were producedso that they were different from each other in the arrangement of areagent layer containing a mediator with respect to a working electrodeor a counter electrode used for Hct measurement, and the responsecurrent and the difference in sensitivity were measured using thesesensors. Also, as sensors according to Comparative Example 1, threetypes of sensors (3-7 to 3-9) were produced so that they were differentfrom each other in the arrangement of a reagent layer containing amediator with respect to a working electrode or a counter electrode usedfor Hct measurement, and the response current and the difference insensitivity were measured using these sensors. The preparation of thespecimens (blood), the measurement of glucose, and the correction of theblood component were carried out in the same manner as in Example 2. Theabove-described respective sensors were produced basically in the samemanner as in Example 2 except for the arrangement of the reagent layer.The reagent layer was produced by dissolving potassium ferricyanide(amount: 60 mM) and taurine (80 mM) in a CMC aqueous solution (0.1 wt %)to prepare a reagent solution, dropping the reagent solution on theelectrodes, and then drying it. The distance between the working,electrode and the counter electrode was set to be at least 0.1 mm.Furthermore, three types of blood samples whose Hct values were adjustedto be 25, 45, and 65, respectively, were provided. With regard to eachof these three blood samples, a current flowing between the electrodesof the sensor when a voltage of 2.5 V was applied for 3 seconds wasmeasured using the sensor, and the response current value and thedifference in sensitivity in the Hct value measurement were determined.FIGS. 7 to 15 show the arrangement patterns of the reagent layers in therespective sensors and the measurement results. In FIGS. 7 to 15, FIGS.7A to 15A show the arrangement pattern of the reagent layer 25, FIGS. 7Bto 15B are graphs each showing changes in response current over timeduring the application of the voltage (V), and FIGS. 7C to 15C aregraphs each showing changes in difference in sensitivity (%) over timeduring the application of the voltage (V). In FIGS. 7 to 15, the samecomponents as those shown in FIGS. 4 to 6 are given the same referencenumerals.

(3-1)

As shown in FIG. 7A, in the sensor of this example, the reagent layer 25was provided so as to extend to the outside of the counter electrode 21used for the Hct measurement, so that the reagent layer 25 was presenton the surface of the counter electrode 21 and at a portion on thecounter electrode side between the electrodes used for the bloodcomponent measurement. The graphs of FIGS. 7B and 7C show the results ofthe measurement of the current flowing between the electrodes of thissensor. As shown in FIGS. 7B and 7C, according to this sensor, thedifference in sensitivity did not depend on the voltage applicationtime, so that the response current reflecting the Hct value could bedetected definitely and favorably.

(3-2)

As shown in FIG. 8A, in the sensor of this example, the reagent layer 25was provided only on the surface of the counter electrode 21. The graphsof FIGS. 8B and 8C show the results of the measurement of the currentflowing between the working electrode 24 and the counter electrode 21 ofthis sensor. As shown in FIGS. 8B and 8C, according to this sensor, thedifference in sensitivity did not depend on the voltage applicationtime, so that the response current reflecting the Hct value could bedetected definitely and favorably.

(3-3)

As shown in FIG. 9A, in the sensor of this example, the reagent layer 25was provided so as to extend to the outside of the counter electrode 21,so that the reagent layer 25 was present on the surface of the counterelectrode 21 and between the electrodes. Note here that no redoxsubstance was present on the working electrode 24. The graphs of FIGS.9B and 9C show the results of the measurement of the current flowingbetween the electrodes of this sensor. As shown in FIGS. 9B and 9C,according to this sensor, the difference in sensitivity did not dependon the voltage application time, so that the response current reflectingthe Hct value could be detected definitely.

(3-4)

As shown in FIG. 10A, in the sensor of this example, the positions ofthe working electrode 24 and the counter electrode 21 that were used forthe Hct measurement were switched so that the reagent layer 25 wasformed on the surface of the counter electrode 21 and at a portion onthe counter electrode side between the electrodes used for the bloodcomponent measurement. The graphs of FIGS. 10B and 10C show the resultsof the measurement of the current flowing between the electrodes of thissensor. As shown in FIGS. 10B and 10C, according to this sensor, thedifference in sensitivity did not depend on the voltage applicationtime, so that the response current reflecting the Hct value could bedetected definitely. However, the difference in sensitivity was slightlysmaller than those exhibited by the sensors according to the examples(3-1), (3-2), and (3-3).

(3-5)

As shown in FIG. 11A, in the sensor of this example, the reagent layer25 was provided so as to extend to the outside of the counter electrode21, so that the reagent layer 25 was present on a part of the surface ofthe counter electrode 21 and at a portion between the electrodes. Thegraphs of FIGS. 11B and 11C show the results of the measurement of thecurrent flowing between the electrodes of this sensor. As shown in FIGS.11B and 11C, according to this sensor, for one second immediately afterthe start of the voltage application (i.e., one second between third tofourth seconds in the drawings), the response current reflecting the Hctvalue could be detected definitely.

(3-6)

As shown in FIG. 12A, in the sensor of this example, the reagent layer25 was provided so as to extend to the outside of the counter electrode21, so that the reagent layer 25 was present on a part of the surface ofthe counter electrode 21. Note here that no redox substance was presentbetween the electrodes. The graphs of FIGS. 12B and 12C show the resultsof the measurement of the current flowing between the electrodes of thissensor. As shown in FIGS. 12B and 12C, according to this sensor, for onesecond immediately after the start of the voltage application (i.e., onesecond between third to fourth seconds in the drawings), the responsecurrent reflecting the Hct value could be detected definitely.

(3-7)

As shown in FIG. 13A, in the sensor of this comparative example, thereagent layer 25 was provided so as to lie over the working electrode24, the counter electrode 21, and the entire region between theseelectrodes. The graphs of FIGS. 13B and 13C show the results of themeasurement of the current flowing between the electrodes of thissensor. As shown in FIGS. 13B and 13C, according to this sensor, theresponse current reflecting the Hct value could not be detecteddefinitely.

(3-8)

As shown in FIG. 14A, in the sensor of this comparative example, thereagent layers 25 were provided so as to lie over the working electrode24 and the counter electrode 21, respectively, and these reagent layers25 were also present at a part of the region between these electrodes.The graphs of FIGS. 14B and 14C show the results of the measurement ofthe current flowing between the electrodes of this sensor. As shown inFIGS. 14B and 14C, according to this sensor, the response currentreflecting the Hct value could not be detected definitely.

(3-9)

As shown in FIG. 15A, in the sensor of this comparative example, thereagent layer 25 was not provided. The graphs of FIGS. 15B and 15C showthe results of the measurement of the current flowing between theelectrodes of this sensor. As shown in FIGS. 15B and 15C, according tothis sensor, the response current reflecting the Hct value could not bedetected.

EXAMPLE 4

In the present example, the response current and the difference insensitivity in the Hct measurement were measured at various appliedvoltages in the range from 0.5 to 6.5 V. The preparation of thespecimens (blood), the measurement of glucose, and the correction of theblood component were carried out in the same manner as in Example 2. Thesensor used for this measurement was produced in the same manner as inExample 3. Note here that the reagent layer 25 was provided on thecounter electrode 21 but not on the working electrode 24 (see FIG. 7A).Furthermore, the response current and the difference in sensitivity weremeasured in the same manner as in Example 3. The results of themeasurement are shown in the graphs of FIGS. 16 to 28. In FIGS. 16 to28, FIGS. 16A to 28A are graphs each showing changes in response current(μA) over time during the application of the voltage (V), and FIGS. 16Bto 28B are graphs each showing changes in difference in sensitivity (%)over time during the application of the voltage (V).

As shown in FIG. 16, even when the applied voltage was 0.5 V, it waspossible to detect the response current reflecting the Hct value.However, as shown in FIGS. 17 to 28, the response current could bemeasured still more definitely when the applied voltage was in the rangefrom 1 to 6.5 V. Furthermore, as shown in FIGS. 17 to 21, the mostpreferable results were obtained when the applied voltage was in therange from 1 to 3 V. When the applied voltage was 5 V or more, thedistortion of the waveform occurred with the passage of time. However,within a short time immediately after the start of the voltageapplication, the response current reflecting the Hct value could bedetected definitely. Although the present example is directed to thecase where the current based on a Hct value was measured with variousapplied voltages under fixed conditions, the present invention is notlimited thereto. It should be noted that even when the applied voltageis outside the range shown in the present example, it is still possibleto detect the response current reflecting the Hct value definitely bysetting other conditions such as the distance between the electrodes andthe amount and the type of the redox substance as appropriate, and theamount of the blood component can be corrected based on thethus-detected Hct value.

EXAMPLE 5

FIG. 30 is a plan view showing still another example of a sensor of thepresent invention. This sensor has an electrode pattern different fromthose of the sensors according to Examples 1 to 4. As shown in FIG. 30,this sensor includes, on an insulating substrate, two electrodes 111 and112 composing a second analysis portion used for Hct measurement on anupstream side and two electrodes 113 and 114 composing a first analysisportion used for blood component measurement on a downstream side withrespect to the flow of blood. In this sensor, reagent layers (not shown)are provided on the first analysis portion and the second analysisportion, respectively. The reagent layer provided on the first analysisportion contains an oxidoreductase such as glucose dehydrogenase and amediator and optionally contains a polymeric material, an enzymestabilizer, and a crystal homogenizing agent, and the arrangementthereof is not particularly limited. On the other hand, the reagentlayer provided on the second analysis portion contains a mediator andoptionally contains a polymeric material. In the second analysisportion, the reagent layer is provided only on the counter electrode.Other than the above, the configuration of the sensor according to thepresent example is the same as that of the sensor according to Example 1or 2.

Next, FIG. 31 shows an example of the configuration of a measuringdevice according to the present invention. For example, the sensor shownin Example 2 can be attached to this measuring device. As shown in FIG.31, this measuring device 130 includes four connectors 137 a to 137 d, aswitching circuit 136, a current/voltage conversion circuit 135, an A/Dconversion circuit 134, a reference voltage source 133, a CPU 131, and aliquid crystal display (LCD) 132 as main components. Note here that thereference voltage source 133 may be grounded. The electrodes 21, 22, 23,and 24 of the sensor are connected to the current/voltage conversioncircuit 135 and the reference voltage source 133 via the connectors 137a to 137 d and the switching circuit 136. The current/voltage conversioncircuit 135 is connected to the CPU 131 via the A/D conversion circuit134.

In this measuring device, measurement of the amount of a blood componentcan be carried out in the following manner, for example.

First, in accordance with an instruction from the CPU 131, the switchingcircuit 136 connects the electrode 21 serving as a working electrode forblood component measurement to the current/voltage conversion circuit135 via the connector 137 a and connects the electrode 22 serving as adetecting electrode for detecting the supply of blood to the referencevoltage source 133 via the connector 137 b. When a constant voltage isapplied between the electrode 21 and the electrode 22 from thecurrent/voltage conversion circuit 135 and the reference voltage source133 in accordance with an instruction from the CPU 131 and blood issupplied to the sensor in this state, a current flows between theelectrodes 21 and 22. This current is converted into a voltage by thecurrent/voltage conversion circuit 135, and the value of this voltage isconverted into a digital value by the A/D conversion circuit 134 and isoutput to the CPU 131. Based on this digital value, the CPU 131 detectsthe supply of the blood.

After the supply of the blood has been detected, the amount of the bloodcomponent is measured. The measurement of the amount of the bloodcomponent is carried out in the following manner, for example. First, inaccordance with an instruction from the CPU 131, the switching circuit136 connects the electrode 21 serving as a working electrode for bloodcomponent measurement to the current/voltage conversion circuit 135 viathe connector 137 a and connects the electrode 23 serving as a counterelectrode for blood component measurement to the reference voltagesource 133 via the connector 137 c.

The current/voltage conversion circuit 135 and the reference voltagesource 133 are turned off, for example, while glucose in the blood isallowed to react with the oxidoreductase for a certain period of time,and after a lapse of a certain period of time, a constant voltage isapplied between the electrodes 21 and 23 in accordance with aninstruction from the CPU 131. A current flows between the electrodes 21and 23, and this current is converted into a voltage by thecurrent/voltage conversion circuit 135. The value of this voltage isconverted into a digital value by the A/D conversion circuit 134 and isoutput to the CPU 131. The CPU 131 converts this digital value to theamount of the blood component.

After the amount of the blood component has been measured, a Hct valueis measured. The measurement of a Hct value is carried out in thefollowing manner, for example. First, in accordance with an instructionfrom the CPU 131, the switching circuit 136 connects the electrode 24serving as a working electrode for Hct measurement to thecurrent/voltage conversion circuit 135 via the connector 137 d andconnects the electrode 21 serving as a counter electrode for Hctmeasurement to the reference voltage source 133.

Then, in accordance with an instruction from CPU 131, a constant voltageis applied between the electrodes 24 and 21 from the current/voltageconversion circuit 135 and the reference voltage source 133. The currentflowing between the electrodes 24 and 21 is converted into a voltage bythe current/voltage conversion circuit 135, and the value of thisvoltage is converted into a digital value by the A/D conversion circuit134 and is output to the CPU 131. The CPU 131 converts the digital valueinto a Hct value.

Using the Hct value and the amount of the blood component obtained inthe above measurements, the amount of the blood component is correctedusing the Hct value with reference to a calibration curve or acalibration curve table prepared previously, and the corrected amount ofthe blood component is displayed in the LCD 132.

Although the present invention has been described with reference to theexamples where glucose is measured, the present invention is not limitedthereto. As already described above, the present invention also isuseful for the measurement of other blood components, such as lacticacid and cholesterol. Moreover, according to the measurement method andthe sensor of the present invention, a current response corresponding tothe type of a sample supplied to the sensor is obtained. This allows thetype of a sample to be identified based on the result obtained.Therefore, according to the measurement method and the sensor of thepresent invention, it is possible to identify, for example, a standardsolution for calibrating the sensor, blood plasma, and blood easily.

INDUSTRIAL APPLICABILITY

As specifically described above, according to a method of measuring ablood component, a sensor used in the method, and a measuring device ofthe present invention, a Hct value can be measured electrochemically andeasily with high accuracy and high reliability and the amount of theblood component can be corrected based on the Hct value. Therefore, themeasurement method, the sensor, and the measuring device of the presentinvention can be used suitably to all the technical fields in which themeasurement of a blood component is required, such as biology,biochemistry, and medical science, and are particularly suitable in thefield of clinical tests.

The invention claimed is:
 1. A method of determining an amount of acomponent in blood using a biosensor comprising at least two electrodesand a reagent layer comprising a mediator and an oxidoreductase, saidreagent layer being disposed on one of said at least two electrodes; themethod comprising: applying a first voltage between said at least twoelectrodes such that the electrode on which the reagent layer isdisposed acts as a working electrode; detecting a first current valuegenerated by the application of the first voltage; applying a secondvoltage between said at least two electrodes such that the electrode onwhich the reagent layer is disposed acts as a counter electrode; anddetecting a second current value generated by the application of thesecond voltage; and calculating the amount of the component using thefirst current value and the second current value, wherein the amount ofthe component is calculated taking into account an amount of hematocritpresent in the blood.
 2. The method of claim 1, wherein the firstvoltage and the second voltage are applied for a time of 0.001 to 10seconds.